Nuclear receptors are a family of transcription factors modulated by small hydrophobic signaling molecules, like steroids, thyroid hormone, free fatty acids, vitamin D and retinoids. Nuclear receptors are important pharmacological targets for drug intervention in disease management. For example, Tamoxifen, an estrogen antagonist, interacts with estrogen receptor to deliver its therapeutic effects on breast cancer; RU486, an antagonist of progesterone receptor, is used for termination of pregnancies and menopause-related disorders; and Dexamethasone interacts with glucocorticoid receptor to suppress immune system function and is useful for treating inflammatory diseases such as asthma.
Nuclear receptors have three independent domains I, II and III. Domains I and III modulate transcriptional activities by interacting with other factors of the transcription complex; Domain II involves in DNA-binding; and Domain III is the ligand-binding domain. Domain II is the most conserved region within the nuclear receptor family, with a unique feature of four pairs of cysteine chelated with two zinc atoms which form a xe2x80x9czinc fingerxe2x80x9d structure. The three domains of nuclear receptors are functionally interchangeable between different members. For example, the androgen receptor DNA-binding domain can be fused to the ligand-binding domain of estrogen receptor and the resulting AR-ER chimeric receptor can modulate androgen-responsive genes by binding to estrogen.
Amino acid sequence homology of the DNA-binding domain between members of nuclear receptor family allows identification of new members of this family through low stringency nucleotide-probe screening. Human genome project also facilitates identification of new genes coding for new nuclear receptors. At present, a few dozens of nuclear receptors have been identified and sequenced, but their ligands have yet to be identified. Recently, a novel nuclear receptor was cloned through degenerate oligonucleotide screening from human and rat cells and was named ubiquitous nuclear receptor (xe2x80x9cURxe2x80x9d), because of its ubiquitous expression pattern in the body. UR has been found to form heterodimers with RXR receptors and binds to double-stranded DNA with the sequence motif: AGGTCANNNNAGGTCA (SEQ ID NO: 1) (xe2x80x9cDR4xe2x80x9d). Promoters containing DR4 can be activated by UR and RXR heterodimer in cultured cells.
LXRa, another new member of the nuclear receptor family has been cloned recently. Amino acid sequence analysis revealed that it shares over 80% homology with UR in the DNA- and ligand-binding domain. The expression of LXRa mRNA is limited to liver and a few other tissues. LXRa has been identified as a transcriptional activator of the cholesterol 7xcex1-hydroxylase gene and plays an important role in cholesterol catabolism.
Recently other nuclear proteins interacting with nuclear receptors have been identified through yeast two-hybrid screening techniques, among which are co-activators and co-repressors of nuclear receptors, e.g., SRC1, 2, 3, and Grip1. These proteins interact with nuclear receptors in a ligand-dependent manner. This property is useful to set up biochemical assays for ligand-receptor interaction.
Steroid derivatives described in this invention are found to modulate the transcriptional activities via binding to UR or LXRa, and thus can be used to treat disorders mediated by such receptors such as atherosclerosis.
An aspect of this invention relates to steroid derivatives of formula (I): 
R3 is hydrogen, amino, carboxyl, oxo, halo, sulfonic acid, xe2x80x94O-sulfonic acid, or alkyl that is optionally inserted with xe2x80x94NHxe2x80x94, xe2x80x94N(alkyl)-, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94Oxe2x80x94SO2xe2x80x94, xe2x80x94SO2xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94SO3xe2x80x94, xe2x80x94SO3xe2x80x94Oxe2x80x94, xe2x80x94COxe2x80x94, xe2x80x94COxe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94COxe2x80x94, xe2x80x94COxe2x80x94NHxe2x80x94, xe2x80x94COxe2x80x94N(alkyl)-, xe2x80x94NHxe2x80x94COxe2x80x94, or xe2x80x94N(alkyl)-COxe2x80x94, and further optionally substituted with hydroxy, halo, amino, carboxyl, sulfonic acid, or xe2x80x94Oxe2x80x940 sulfonic acid. Each of R1, R2, R4, R4xe2x80x2, R6, R7,R11, R12, R15, R16 and R17xe2x80x2, independently, is hydrogen, hydroxy, amino, carboxyl, oxo, halo, sulfonic acid, xe2x80x94O-sulfonic acid, or alkyl that is optionally inserted with xe2x80x94NHxe2x80x94, xe2x80x94N(alkyl)-, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94Oxe2x80x94SO2xe2x80x94, xe2x80x94SO2xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94SO3xe2x80x94, xe2x80x94SO3xe2x80x94Oxe2x80x94, xe2x80x94COxe2x80x94, xe2x80x94COxe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94COxe2x80x94, xe2x80x94COxe2x80x94NHxe2x80x94, xe2x80x94COxe2x80x94N(alkyl)-, xe2x80x94NHxe2x80x94COxe2x80x94, or xe2x80x94N(alkyl)-COxe2x80x94, and further optionally substituted with hydroxy, halo, amino, carboxyl, sulfonic acid, or xe2x80x94O-sulfonic acid. Each of R5, R8, R9, R10, R13, and R14, independently, is hydrogen, alkyl, haloalkyl, hydroxyalkyl, alkoxy, hydroxy, or amino. R17 is xe2x80x94Xxe2x80x94Yxe2x80x94Z. X is a bond, or alkyl or alkenyl, optionally inserted with xe2x80x94NHxe2x80x94, xe2x80x94N(alkyl)-, xe2x80x94Oxe2x80x94, or xe2x80x94Sxe2x80x94, and further optionally forming a cyclic moiety with R16 and the 2 ring carbon atoms to which R16 and R17 are bonded. Y is xe2x80x94COxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94Oxe2x80x94SO2xe2x80x94, xe2x80x94SO2xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94SO3xe2x80x94, xe2x80x94SO3xe2x80x94Oxe2x80x94, xe2x80x94COxe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94COxe2x80x94, xe2x80x94COxe2x80x94NHxe2x80x94, xe2x80x94COxe2x80x94N(alkyl)-, xe2x80x94NHxe2x80x94COxe2x80x94, xe2x80x94N(alkyl)-COxe2x80x94, or a bond. Z is alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl, and is optionally substituted with hydroxy, alkoxy, amino, halo, sulfonic acid, xe2x80x94O-sulfonic acid, carboxyl, oxo, alkyloxycarbonyl, alkylcarbonyloxy, alkylaminocarbonyl, alkylcarbonylamino, alkylcarbonyl, alkylsulfinyl, alkylsulfonyl, or alkylthio; or is xe2x80x94CH(A)xe2x80x94B. A being a side chain of an amino acid, and B is hydrogen, xe2x80x94NRaRb, or xe2x80x94COORc wherein each of Ra, Rb, and Rc, independently, is hydrogen or alkyl. n is 0, 1, or 2. Note that when Z is substituted with carboxyl or alkyloxycarbonyl, Y is a bond and either X or Z contains at least one double bond, and that when Y is a bond, either X is xe2x80x94NH-alkyl-, xe2x80x94NH-alkenyl-, xe2x80x94N(alkyl)-alkyl-, xe2x80x94N(alkyl)-alkenyl-, xe2x80x94O-alkyl-, xe2x80x94O-alkenyl-, xe2x80x94S-alkyl-, or xe2x80x94S-alkenyl-; or Z is substituted with halo, sulfonic acid, xe2x80x94O-sulfonic acid, alkylsulfinyl, or alkylsulfonyl, or is alkenyl.
Another aspect of this invention relates to steroid derivatives having the formula (I) as depicted above. Each of R1, R2, R3, R4, R4xe2x80x2, R6, R7, R11, R12, R15, R16, and R17xe2x80x2, independently, is hydrogen, hydroxy, amino, carboxyl, oxo, halo, sulfonic acid, xe2x80x94O-sulfonic acid, or alkyl that is optionally inserted with xe2x80x94NHxe2x80x94, xe2x80x94N(alkyl)-, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94Oxe2x80x94SO2xe2x80x94, xe2x80x94SO2xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94SO3xe2x80x94, xe2x80x94SO3xe2x80x94Oxe2x80x94, xe2x80x94COxe2x80x94, xe2x80x94COxe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94COxe2x80x94, xe2x80x94COxe2x80x94NHxe2x80x94, xe2x80x94COxe2x80x94N(alkyl)-, xe2x80x94NHxe2x80x94COxe2x80x94, or xe2x80x94N(alkyl)-COxe2x80x94, and further optionally substituted with hydroxy, halo, amino, carboxyl, sulfonic acid, or xe2x80x94O-sulfonic acid. Each of R5, R8, R9, R10, R13, and R14, independently, is hydrogen, alkyl, haloalkyl, hydroxyalkyl, alkoxy, hydroxy, or amino. R17 is xe2x80x94Xxe2x80x94Yxe2x80x94Z. X is a bond, or alkyl or alkenyl, optionally inserted with xe2x80x94NHxe2x80x94, xe2x80x94N(alkyl)-, xe2x80x94Oxe2x80x94, or xe2x80x94Sxe2x80x94, and further optionally forming a cyclic moiety with R16 and the 2 ring carbon atoms to which R16 and R17 are bonded. Y is xe2x80x94COxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94Oxe2x80x94SO2xe2x80x94, xe2x80x94SO2xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94SO3xe2x80x94, xe2x80x94SO3xe2x80x94Oxe2x80x94, xe2x80x94COxe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94COxe2x80x94, xe2x80x94COxe2x80x94NHxe2x80x94, xe2x80x94COxe2x80x94N(alkyl)-, xe2x80x94NHxe2x80x94COxe2x80x94, xe2x80x94N(alkyl)xe2x80x94CO-, or a bond. Z is alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl, and is optionally substituted with hydroxy, alkoxy, amino, halo, sulfonic acid, xe2x80x94O-sulfonic acid, carboxyl, oxo, alkyloxycarbonyl, alkylcarbonyloxy, alkylaminocarbonyl, alkylcarbonylamino, alkylcarbonyl, alkylsulfinyl, alkylsulfonyl, or alkylthio; or is xe2x80x94CH(A)xe2x80x94B. A is an amino acid side chain containing an aromatic moiety, and B is hydrogen, xe2x80x94NRaRb, or xe2x80x94COORc wherein each of Ra, Rb, and Rc, independently, is hydrogen or alkyl. n is 0, 1, or 2. Note that when Z is substituted with carboxyl or alkyloxycarbonyl, Y is a bond and either X or Z contains at least one double bond, and that when Y is a bond, either X is xe2x80x94NH-alkyl-, xe2x80x94NH-alkenyl-, xe2x80x94N(alkyl)-alkyl-, xe2x80x94N(alkyl)-alkenyl-, xe2x80x94O-alkyl-, xe2x80x94O-alkenyl-, xe2x80x94S-alkyl-, or xe2x80x94S-alkenyl-; or Z is substituted with halo, sulfonic acid, xe2x80x94O-sulfonic acid, alkylsulfinyl, or alkylsulfonyl, or is alkenyl.
A further aspect of this invention relates to steroid derivatives of formula (I), supra. Each of R1, R2, R3, R4, R4xe2x80x2, R6, R7, R11, R12, R15, R16, and R17xe2x80x2, independently, is hydrogen, hydroxy, amino, carboxyl, oxo, halo, sulfonic acid, xe2x80x94O-sulfonic acid, or alkyl optionally inserted with xe2x80x94NHxe2x80x94, xe2x80x94N(alkyl)-, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94Oxe2x80x94SO2xe2x80x94, xe2x80x94SO2xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94SO3xe2x80x94, xe2x80x94SO3xe2x80x94Oxe2x80x94, xe2x80x94COxe2x80x94, xe2x80x94COxe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94COxe2x80x94, xe2x80x94COxe2x80x94NHxe2x80x94, xe2x80x94COxe2x80x94N(alkyl)-, xe2x80x94NHxe2x80x94COxe2x80x94, or xe2x80x94N(alkyl)-COxe2x80x94, and further optionally substituted with hydroxy, halo, amino, carboxyl, sulfonic acid, or xe2x80x94O-sulfonic acid. Each of R5, R8, R9, R10, R13, and R14, independently, is hydrogen, alkyl, haloalkyl, hydroxyalkyl, alkoxy, hydroxy, or amino. R17 is xe2x80x94Xxe2x80x94Yxe2x80x94Z. X is a bond, or alkyl or alkenyl, optionally inserted with xe2x80x94NHxe2x80x94, xe2x80x94N(alkyl)-, xe2x80x94Oxe2x80x94, or xe2x80x94Sxe2x80x94, and further optionally forming a cyclic moiety with R16 and the 2 ring carbon atoms to which R16 and R17 are bonded. Y is xe2x80x94COxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94Oxe2x80x94SO2xe2x80x94, xe2x80x94SO2xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94SO3xe2x80x94, xe2x80x94SO3xe2x80x94Oxe2x80x94, xe2x80x94COxe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94COxe2x80x94, xe2x80x94COxe2x80x94NHxe2x80x94, xe2x80x94COxe2x80x94N(alkyl)-, xe2x80x94NHxe2x80x94COxe2x80x94, xe2x80x94N(alkyl)-COxe2x80x94, or a bond. Z is alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl, and is optionally substituted with hydroxy, alkoxy, amino, halo, sulfonic acid, xe2x80x94O-sulfonic acid, carboxyl, oxo, alkyloxycarbonyl, alkylcarbonyloxy, alkylaminocarbonyl, alkylcarbonylamino, alkylcarbonyl, alkylsulfinyl, alkylsulfonyl, or alkylthio; or is xe2x80x94CH(A)xe2x80x94B. A is a side chain of an amino acid, and B is hydrogen, xe2x80x94NRaRb, or xe2x80x94COORc wherein each of Ra, Rb, and Rc, independently, is hydrogen or alkyl. n is 0, 1, or 2. Note that when Z is substituted with carboxyl or alkyloxycarbonyl, Y is a bond and either X or Z contains at least one double bond, and that when Y is a bond, either X is xe2x80x94NH-alkyl-, xe2x80x94NH-alkenyl-, xe2x80x94N(alkyl)-alkyl-, xe2x80x94N(alkyl)-alkenyl-, xe2x80x94O-alkyl-, xe2x80x94O-alkenyl-, xe2x80x94S-alkyl-, or xe2x80x94S-alkenyl-; or Z is substituted with halo, sulfonic acid, xe2x80x94O-sulfonic acid, alkylsulfinyl, or alkylsulfonyl, or is alkenyl; and that at least one of R3 and R4, R4 and R5, R5 and R6, R7 and R8, R12 and R13, and R15 and R16 independently, is deleted to form a double bond. One subset of the just-described steroid derivatives encompasses compounds which are featured by the presence of at least one double bond in the rings, which are formed by deleting one or more of the following pairs of substituents: R3 and R4, R4 and R5, R12 and R13, and R15 and R16. Another subset encompasses compounds which are featured by that Z is alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl, and optionally substituted with hydroxy, alkoxy, amino, or halo; or is xe2x80x94CH(A)xe2x80x94B. A and B are as described above.
Note that X and Z optionally join together to form a cyclic moiety. For example, if both X and Z are alkyl, and Y is xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94, a lactone results from joining X and Z.
A salt of the steroid derivative of this invention is also within the scope of this invention and can be formed, for example, between the steroid of this invention having a carboxylate and a cationic counterion such as an alkali metal cation, e.g., a sodium ion or a potassium ion; or an ammonium cation that can be substituted with organic groups, e.g., a tetramethylammonium ion or a diisopropyl-ethylammonium ion. A salt of this invention can also form between the steroid derivative of this invention having a protonated amino group and an anionic counterion, e.g., a sulfate ion, a nitrate ion, a phosphate ion, or an acetate ion.
Set forth below are some examples of steroid derivatives of this invention: 
As used herein, the term xe2x80x9calkylxe2x80x9d in this disclosure denotes a straight or branched hydrocarbon chain containing 1-8 carbon atoms. Some examples of an alkyl group are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, or 2-methylpentyl. By the term xe2x80x9ccycloalkylxe2x80x9d is meant a cyclic hydrocarbon chain that has 3-8 carbon atoms. The cycloalkyl groups herein described may also contain fused rings. Fused rings are rings that share a common carbon-carbon bond. Examples of cycloalkyl groups include, but not limited to, cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, and norbornyl.
The term xe2x80x9calkenylxe2x80x9d refers to a straight or branched hydrocarbon chain containing 2-8 carbon atoms and characterized in having one or more double bonds. Examples of a typical alkenyl include, but not limited to, allyl, propenyl, 2-butenyl, 3-hexenyl and 3-octenyl groups. The term xe2x80x9ccycloalkenylxe2x80x9d is meant a cyclic hydrocarbon chain containing 3-8 carbon atoms and having at least one or more double bonds. Similar to the definition of cycloalkyl groups above, cycloalkenyl groups may also contain fused rings. Some examples of cycloalkenyl groups are cyclopentenyl, cyclohexenyl, cycloheptenyl, norbornylenyl, and cyclooctenyl groups.
The term xe2x80x9calkynylxe2x80x9d refers to a straight or branched hydrocarbon chain containing 2-8 carbon atoms and characterized in having one or more triple bonds. Some examples of a typical alkynyl are ethynyl, 2-propynyl, and 3-methylbutynyl.
The terms xe2x80x9cheterocycloalkylxe2x80x9d and xe2x80x9cheterocycloalkenylxe2x80x9d refer to cycloalkyl and cycloalkenyl groups which contain one or more heteroatoms, such as, nitrogen, oxygen, or sulfur. Typical heterocycloalkyl and heterocycloalkenyl groups include tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino, pyrrolinyl and pyrrolidinyl.
xe2x80x9cArylxe2x80x9d represents an aromatic moiety which contains 6-12 carbon atoms and can contain fused rings. A fused ring is an aromatic group which contains at least two aryl rings sharing a common carbon-carbon bond. Typical examples of aryl include phenyl and naphthyl.
xe2x80x9cHeteroarylxe2x80x9d groups in this disclosure are aromatic groups containing 5 to 12 ring atoms, in which one or more of these ring atoms are heteroatoms as defined above. Some examples of heteroaryl groups are pyridyl, pyrazinyl, furyl, pyrrolyl, thienyl, thiazolyl, benzimidazolyl, and imidazolyl.
The positions of substituents on each of the cyclic groups described herein may be at any available position, unless specified otherwise. For example, a methyl substituent on a benzene ring can be attached at the ortho, meta, or para position.
The term xe2x80x9calkoxyxe2x80x9d is defined as the moiety xe2x80x9cxe2x80x94O-alkyl.xe2x80x9d Some examples are methoxy, ethoxy, propoxy, isopropoxy, and t-butoxy. xe2x80x9cHaloxe2x80x9d represents a halogen atom, such as, fluoro, chloro, bromo, or iodo. By the terms xe2x80x9chaloalkylxe2x80x9d and xe2x80x9chydroxyalkylxe2x80x9d are meant alkyl groups which are respectively substituted with one or more halogen atoms and one or more hydroxy groups. The nitrogen atom in an amino or amido group present in a steroid derivative of this invention can be mono- or di-substituted with an alkyl, a cycloalkyl, a heterocycloalkyl, an aryl, or a heteroaryl.
For convenience, a divalent moiety is named herein as if it were a monovalent moiety. For example, xe2x80x9calkyl,xe2x80x9d such as CH3, which is assigned to X, actually stands for xe2x80x9calkylene,xe2x80x9d such as xe2x80x94CH2xe2x80x94. As recognized by a skilled person in the art, steroid derivatives described herein contain stereocenters. Both the racemic mixtures of isomers and the optically pure isomers are within the scope of this invention.
Yet another aspect of this invention relates to a pharmaceutical composition for treating a UR- or LXRa-mediated disorder which contains a pharmaceutically acceptable carrier and an effective amount of one or more of the steroid derivatives described above. The use of such a steroid derivative or a salt thereof for the manufacture of a medicament for treating the above-mentioned disorders is also within the scope of this invention.
A still further aspect of this invention relates to a pharmacological composition for treating cancer, including solid tumors and leukemia, and immune dysfunction. The pharmacological composition contains a pharmaceutically acceptable carrier and an effective amount of one or more of a steroid derivative of formula (I), supra. Each of R1, R2, R3, R4, R4xe2x80x2, R6, R7, R11, R12, R15, R16, and R17xe2x80x2, independently, is hydrogen, hydroxy, amino, carboxyl, oxo, halo, sulfonic acid, xe2x80x94O-sulfonic acid, or alkyl that is optionally inserted with xe2x80x94NHxe2x80x94, xe2x80x94N(alkyl)-, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94Oxe2x80x94SO2xe2x80x94, xe2x80x94SO2xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94SO3xe2x80x94, xe2x80x94SO3xe2x80x94Oxe2x80x94, xe2x80x94COxe2x80x94, xe2x80x94COxe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94COxe2x80x94, xe2x80x94COxe2x80x94NHxe2x80x94, xe2x80x94COxe2x80x94N(alkyl)-, xe2x80x94NHxe2x80x94COxe2x80x94, or xe2x80x94N(alkyl)-COxe2x80x94, and further optionally substituted with hydroxy, halo, amino, carboxyl, sulfonic acid, or xe2x80x94O-sulfonic acid. Each of R5, R8, R9, R10, R13, and R14, independently, is hydrogen, alkyl, haloalkyl, hydroxyalkyl, alkoxy, hydroxy, or amino. R17 is xe2x80x94Xxe2x80x94Yxe2x80x94Z, in which X is a bond, or alkyl or alkenyl, optionally inserted with xe2x80x94NHxe2x80x94, xe2x80x94N(alkyl)-, xe2x80x94Oxe2x80x94, or xe2x80x94Sxe2x80x94, and further optionally forming a cyclic moiety with R16 and the 2 ring carbon atoms to which R16 and R are bonded; Y is xe2x80x94COxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94Oxe2x80x94SO2xe2x80x94, xe2x80x94SO2xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94SO3xe2x80x94, xe2x80x94SO3xe2x80x94Oxe2x80x94, xe2x80x94COxe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94COxe2x80x94, xe2x80x94COxe2x80x94NHxe2x80x94, xe2x80x94COxe2x80x94N(alkyl)-, xe2x80x94NHxe2x80x94COxe2x80x94, xe2x80x94N(alkyl)-COxe2x80x94, or a bond; and Z is alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl, and is optionally substituted with hydroxy, alkoxy, amino, halo, sulfonic acid, xe2x80x94O-sulfonic acid, carboxyl, alkyloxycarbonyl, alkylcarbonyloxy, alkylaminocarbonyl, alkylcarbonylamino, alkylcarbonyl, alkylsulfinyl, alkylsulfonyl, or alkylthio; or is xe2x80x94CH(A)xe2x80x94B with A being a side chain of an amino acid, and B being hydrogen, NRaRb, or xe2x80x94COORc wherein each of Ra, Rb, and Rc, independently, is hydrogen or alkyl; and n is 0, 1, or 2. When Z is substituted with carboxyl, Y is a bond and either X or Z contains at least one double bond, and when Y is a bond, either X is xe2x80x94NH-alkyl-, xe2x80x94NH-alkenyl-, xe2x80x94N(alkyl)-alkyl-, xe2x80x94N(alkyl)-alkenyl-, xe2x80x94O-alkyl-, xe2x80x94O-alkenyl-, xe2x80x94S-alkyl-, or xe2x80x94S-alkenyl-; or Z is substituted with halo, sulfonic acid, xe2x80x94O-sulfonic acid, alkylsulfinyl, or alkylsulfonyl, or is alkenyl. The use of a just-described steroid derivative or a salt thereof for the manufacture of a medicament for treating the above-mentioned disorders is also within the scope of this invention.
Still another aspect of the present invention relates to a method of treating a UR- or LXRa-mediated disorder by administering to a patient in need thereof an effective amount of one of the pharmaceutical compositions decribed above. Some examples of UR- or LXRa-mediated disorders are: liver cirrhosis, gallstone disease, hyperlipoproteinemias, Alzheimer""s disease, anemia, chronic inflammatory diseases (e.g., rheumatoid arthritis), metabolic disorders (e.g., diabetes), and cancers which are associated with UR expression, e.g., breast cancer, colon cancer, prostate cancer, and leukemia. Patients with other disorders such as atherosclerosis and liver cholestasis can also be treated with one of the pharmaceutical compositions described above.
Other features or advantages of the present invention will be apparent from the following detailed description of several embodiments, and also from the appending claims.
A steroid derivative of this invention can be prepared by forming an amide bond between a steroid having a C17 carboxyl-containing substituent and an amino-containing compound or between a steroid having a C17 amino-containing substituent and a carboxyl-containing compound. Similarly, an ester bond can be formed between a steroid with a C17 carboxyl-containing substituent and a hydroxyl-containing compound, or between a steroid with a C17 hydroxyl-containing substituent and a carboxyl-containing compound. Some examples of a steroid that can be used as a starting material are cholic acid (e.g., ursodeoxycholic acid, hyocholic acid, and hyodeoxycholic acid), androstan-17-carboxylic acid (e.g., androstan-3-oxo-17-carboxylic acid and d5-androsten-3-ol-17-carboxylic acid) and pregnan-20-ol (e.g., d5-pregnen-3,17-diol or pregnan-17-ol-3-one). Synthesis of these steroids can be found in the literature, e.g., Roda A. et al., F. Lipid Res. vol. 35, pages 2268-2279 (1994) and Roda A. et al., Dig. Dis. Sci. vol. 34, pages 24S-35S (1987). Some examples of compounds that can be used to couple to a steroid to form a steroid derivative of this invention are aniline, glycine, phenylalanine, or benzoic acid. Examples of a coupling reagent that can be used in the amide- or ester-forming reaction include 1-ethyl-3-[3-dimethylaminopropyl]-carbodiimide (EDC), dicyclohexyl-carbodiimide (DCC), N-hydroxybenzo-triazole (HOBt), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-phosphate (HBTU), or benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP). The amide- or ester-forming reaction can take place in any solvents that are suitable with the starting materials and reagents. Note that if the reaction takes place in an aqueous solvent, e.g., a buffered solution (or in combination with other miscible organic solvents such as alcohol), isolation of the steroid product for in vitro or in vivo screening assays is not necessary, as the product is already in suitable assaying conditions, i.e., in an aqueous buffered medium. Protection of functional groups, e.g., hydroxyl or keto, on the steroids is not needed. See, e.g., Example 1 below. Due to the simplicity of the reaction, it can be easily automated. Isolation and quantification of the product can be done by thin-layer chromatography, high pressure liquid chromatography, gas chromatography, capillary electrophoresis, or other analytical and preparative procedures. Trifluoromethyl- and taurine-conjugated steroid derivatives can be prepared according to methods described in Li, S. et al., Chem. Phys. Lipids 99:33-71 (1999) and Kurosawa, T. et al., Steroids, 60:439-444 (1995), respectively. As to the preparation of 3xcex2-hydroxy-5-cholesten-25(R)-26-carboxylic acid derivatives, see Kim, H. et al., J. Lipid Res. 30:247 (1989) and Varma, R. K. et al., J. Org. Chem. 40:3680 (1975). Steroid derivatives having a side chain that contains a double bond, e.g., between C24 and C25, can be prepared according to the following scheme: 
3-beta-t-butyldimethylsilyloxy-delta[5]-cholen-24-al and 3-alpha,6-alpha-di(t-butyldimethylsilyloxy)5-beta-cholan-24-al were prepared using NaBH4 and pyridinium chlorochromate according to methods described in Somanathan et al., Steroids 43:651-655 (1984). Ethyl-3-beta-t-butyldimethylsilyloxy-delta[5,24]-cholestenoate and ethyl-3a,6a-di(t-butyldimethylsilyloxy)-delta[24]-cholestanoate were then prepared via Wittig-Horner reaction using triethyl 2-phosphono-propionate and a suitable base according to methods described in Lund et al., Arterioscler. Thromb. Vasc. Biol. 16:208-212 (1996). After the t-butyldimethylsilyloxyl groups were removed, ethyl ester groups were hydrolyzed under alkaline conditions.
As mentioned above, a pharmaceutical composition containing a steroid derivative or a salt of this invention in an effective amount can be used to treat UR- or LXRa-mediated disorders. Also within the scope of this invention is a method of treating a UR- or LXRa-mediated disorder such as astherosclerosis by administering to a patient such a composition. An effective amount is defined as the amount of the derivative which, upon administration to a patient in need, confers a therapeutic effect on the treated patient. The effective amount to be administered to a patient is typically based on body surface area, patient weight, and patient condition. The interrelationship of dosages for patients (based on milligrams per meter squared of body surface) is described by Freireich et al., Cancer Chemother. Rep. 1966, 50, 219. Body surface area may be approximately determined from height and weight of the patient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardley, N.Y., 1970, 537. An effective amount of a compound of this invention used to practice the invention can range from about 1 mg/kg to about 2 g/kg, e.g., from about 1 mg/kg to about 1 g/kg, or from about 1 mg/kg to about 500 mg/kg. Effective doses will also vary, as recognized by those skilled in the art, dependant on route of administration, excipient usage, and the possibility of co-usage with other therapeutic treatments.
The pharmaceutical composition may be administered via the parenteral route, including subcutaneously, intraperitoneally, intramuscularly and intravenously. Examples of parenteral dosage forms include aqueous solutions of the active agent, in a isotonic saline, 5% glucose or other well-known pharmaceutically acceptable excipient. Solubilizing agents such as cyclodextrins, or other solubilizing agents well-known to those familiar with the art, can be utilized as pharmaceutical excipients for delivery of the therapeutic compounds.
The steroid derivatives of this invention can also be formulated into dosage forms for other routes of administration utilizing well-known methods. They can be formulated, for example, in dosage forms for oral administration in a gel seal, a syrup, a capsule, or a tablet. Capsules may comprise any well-known pharmaceutically acceptable material such as gelatin or cellulose derivatives. Tablets may be formulated in accordance with the conventional procedure by compressing mixtures of the compound of this invention and a solid carrier, and a lubricant. Examples of solid carriers include starch and sugar bentonite. The steroid derivatives of this invention can also be administered in a form of a hard shell tablet or a capsule containing a binder (e.g., lactose or mannitol) and a conventional filler.
The level of interaction between the UR or LXRa protein and a steroid derivative of this invention can be preliminarily evaluated using various assays as described below:
Protease protection assay is a simple assay for measuring the level of interaction between a test steroid and the UR or LXRa protein. This assay can be done by using a 35S-Met radiolabeled rat UR or human LXRa protein. The radiolabeled protein is then incubated with the steroid of this invention and digested with a protease, e.g., trypsin. A control experiment is done by incubating UR receptor with a protease but without the steroid. Protein fragments from both assays are electrophoresed on a polyacrylamide gel. The fragments from each of the assays can be visualized by exposing the gel to X-ray films and compared side-by-side. A test steroid, if binds to the UR or LXRa protein, will protect the receptor from being digested by the protease. As a result, reactions that result in binding between the steroid and UR will lead to fewer bands of low molecular weights than those that do not result in binding between the two molecules.
The co-activator binding assay employs a fusion protein formed between a glutathione S-transferase (GST) and a co-activator of UR, e.g., Grip1. The GST moiety of the fusion protein binds to a glutathione-coated solid support, thereby immobilizing the fusion protein. UR and a steroid of this invention are then incubated with the immobilized fusion protein. Subsequently, any bound UR is released and collected from the solid support. The proteins are then electrophoresed on a polyacrylamide gel and visualized by exposing the gel to X-ray films. If the steroid interacts with UR, less UR will bind to the fusion protein, and a lighter band would therefore result on the gel. By monitoring the intensity of the band of the bound UR, one can estimate the binding of the steroid to UR.
Yeast two-hybrid binding assay is a sensitive assay for identifying UR modulating compounds by monitoring transcriptional activation. General descriptions of these assay can be found in, e.g., Chien C. T. et al., Proc. Natl. Acad. Sci. USA, vol. 88, 9578-9582 (1991); Fields, S. et al., Nature, vol. 340, 245-247 (1989); and Green, M. B. et al., Curr. Biol., vol. 2, 403-405 (1992). In this screening method, a steroid of this invention that modulates the interaction of UR or LXRa with its natural ligand will have an effect on the transcriptional activation of a reporter gene. In a specific assay, two plasmids are introduced into a yeast cell. One expresses a fusion protein having a GAL4 DNA binding domain and a UR natural ligand, and the other expresses a fusion protein containing a UR ligand binding domain and a GAL4 activation domain. If the steroid interacts with UR and disrupts the binding of UR to its natural ligand, the activity of the reporting gene (Gal4) will be altered. The changes in reporter activities (i.e., xcex2-galactosidase activities) can be measured with a commercial luminescence kit.
Mammalian cell transfection can also be used to screen steroid derivatives that affect the interaction between the UR protein and a steroid of this invention. A rat UR or human LXR gene and a human RXRa gene are cloned into a mammalian expression vector (e.g., pSG5 from Strategene) and overexpressed. A heterologous promoter is formed by inserting four tandem repeats of a hormone response element DR4 into the vector upstream to a c-fos promoter sequence, which is followed by a sequence encoding luciferase. The entire construct is named DR4-fos-luc. DR4-fos-luc is then co-transfected with pSG5/rUR or CMV/hLXR and pSG5/hRXRa into mammalian cells, e.g., COS-1 cells. An ethanol solution containing a steroid of this invention is then added to the transfected cells. The steroid, if interacts to the UR or LXRa protein, affects the level at which the luciferase gene is activated. The cells are then lysed and assayed for luciferase activity with a commercial assay kit and a luminometer. A high intensity of luminescence indicates that the steroid is a potent UR or LXR agonist.
Another chimeric receptor that can be used in this assay is constructed by fusing oligonucleotides encoding the ligand-binding domain of rat UR to a human AR gene lacking ligand-binding site coding region. For this chimeric receptor, a reporter gene ARE-fos-luc is constructed by inserting three tandem repeats of Androgen Response Element (ARE) into the vector upstream to a c-fos promoter which is followed by a luciferase reporter gene. After adding a steroid of this invention to the medium of the transfected cells, the steroid can interact with UR and affect the level of activation of ARE-fos-luc in cultured cells. The level of luminescence activity thus indicates the level of UR modulation by the steroid.
Yet another assay involves expressing rUR gene in PC-3 cells by retroviral infection. See Underwood et al., J. Biol. Chem., vol. 273, pages 4266-4274 (1998). The transfected cells are then seeded in media containing delipidated serum and then treated with a solution containing a steroid of this invention. The PC-3 cells are later washed with phosphate buffered saline (PBS) and treated with 100 mg/ml amphotericin B in DMEM media without serum at 37EC. Amphotericin B functions to kill cells containing cholesterol in the cell membrane. The cells are then fixed in 10% TCA and stained with Sulforhodamine B after more washing. Viable cells are stained and can then be assessed using a colorimetric assay. The amount of dye is directly proportional to number of surviving cells on the culture plates. From comparing the number of viable cells between assays with and without a steroid, one can estimate the effect the steroid has on the de novo synthesis of cholesterol.
A still further assay makes use of nitrogen monoxide (NO) as an indicator of the level of inflammation. Cells from a murine macrophage cell line RAW264.7 are incubated with a steroid of this invention for 24 hours. The macrophages are then activated by adding lipopolysaccharide (LPS) and gamma-interferon. The NO production of activated macrophages can be monitored indirectly by quantifying NO2 in the media according to Green L. et al., Anal. Biochem., vol. 126, 131-138 (1982). The reduced amount of NO in comparison to that of a control experiment in which no steroid is used indicates that the steroid used in the assay has inhibitory effect on inflammation.
Using the same murine macrophage cell line RAW264.7, constitutive expression of rat UR and human RXRa gene by retroviral systems transforms these cells into foam-cell-like morphology and integrated into clumps while increasing cell sizes and undergo apoptosis. Foam cells originated from macrophages are the major components in pathological plaque which is usually found on the inner wall of blood vessels in patients suffering from atherosclerosis. Steroid derivatives of this invention which modulate UR can suppress the progression of macrophage-foam cell transformation at different stages, and can be used in the treatment or prevention of atherosclerosis. See Kellner-Weibel et al., Arterioscler. Thromb. Vasc. Biol., vol. 18, pages 423-431 (1998).
Yet another assay measures the effect of a steroid of this invention has on the level of adipocyte differentiation on fibroblasts. Specifically, the level of adipocyte differentiation in murine fibroblasts 3T3-L1 containing rat UR gene at sub-confluent conditions is measured. Constitutive expression of rat UR gene in murine fibroblasts 3T3-L1 can be done by using retroviral systems. Full-length rat UR cDNA are inserted into retroviral expression vector MV7. Infected 3T3-L1 cells that are G418-resistant are treated with insulin, dexamethacine, and 1-methyl-3-isobutylxanthine (MIX) to induce adipocyte differentiation. A control experiment can be done by inserting human UR cDNA into MV7 in the antisense orientation. Cells infected with hUR-antisense constructs and parent 3T3-L1 cells are also treated with the same insulin cocktail under same cell density. Cells infected with rUR are shown to accumulate more Red oil O positive lipid drops than parent cells, while cells infected with hUR antisense are shown to have less Red oil O positive lipid drops. Thus, the finding shows that the expression of UR in fibroblasts plays a role in adipocyte differentiation.